We have constructed a mammary gland expression vector that contained the goat β-casein gene pro-moter, 5’upstream regulatory region, exons 1, 2, intron 1 as well as the human serum albumin (hALB) mini-gene (includin...We have constructed a mammary gland expression vector that contained the goat β-casein gene pro-moter, 5’upstream regulatory region, exons 1, 2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F0 mice were obtained. Of the 33, 8 mice (5 , 3 ) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.展开更多
Due to the functional importance of bovine milk protein polymorphisms, their correct discrimination is of great interest both from a scientific and practical point of view. Nowadays a large number of commercial platfo...Due to the functional importance of bovine milk protein polymorphisms, their correct discrimination is of great interest both from a scientific and practical point of view. Nowadays a large number of commercial platforms are available for semiautomated or fully automated SNP geno-typing. However, in some cases the use of simple and rather cheap methods is an effective tool to be implemented within one’s own laboratory for the routine analysis of a specific SNP. The present paper describes two simple tests based on the bidirectional allele-specific polymerase chain reaction (BAS-PCR) developed for the identification of β-casein (CSN2) B and I genetic variants. The practical application of the two methods on a panel of 84 Italian Brown bulls and 100 Italian Friesian cows is also discussed, including the biological significance of the two genetic variants and the importance of taking their occurrence into account when linkage analyses are performed on milk functional properties. A combined system for analysing milk protein variants by isoelectrofocusing (IEF) and the BAS-PCR assay developed for CSN2*I is described.展开更多
Cationic peptide with the sequence INKKI 41-45 was isolated from bovine β-casein after tryptic hydrolysis and synthetized. The aim of this work was to evaluate the antiproliferative activity in vitro and antitumor ef...Cationic peptide with the sequence INKKI 41-45 was isolated from bovine β-casein after tryptic hydrolysis and synthetized. The aim of this work was to evaluate the antiproliferative activity in vitro and antitumor effect in animal model. The in vitro cytotoxicity was evaluated on B16F10 melanoma cells by MTT assay. Detection of apoptosis was measured using the annexin V/PI double staining and cell cycle analysis performed flow cytometry. Caspase-3 activity was analyzed with substrate specific fluorogenic DEVD-MCA. In vivo, antitumor activity was evaluated in B16F10 melanoma tumor-bearing C57BL/6J mice. The animals were treated with 55 mg/kg INKKI administered into peritumoral region, while control group received saline solution. The following antitumor parameters were examined: tumor volume, number of metastases, tumor delayed time, tumor doubling time. Histological analyses were performed with H & E staining. The results showed that INKKI induced dose-response cytotoxicity selective for B16F10 melanoma cells (IC50 1.7 μM) and did not present cytotoxic effects for FN1 fibroblast cells. INKKI-induced apoptosis detected trough of annexin V/PI assay and it was accompanied with an increase of sub-G1 apoptotic fractions and significant increase of caspase-3 cleavage. The tumor-bearing mice treated with INKKI showed a significant reduction in tumor volume of 72.62% and decreased of metastasis number loci. In addition, INKKI caused a significant delay in tumor growth and prolonged the tumor doubling time. Histological analysis revealed an increased of necrosis areas and reduction of tumor cells in tumor treated with INKKI, it was a many hallmark of its antitumor effects observed from in vivo experiments. In conclusion, we show that INKKI is a peptide that could be considered a new putative candidate development to anticancer therapy drug.展开更多
文摘We have constructed a mammary gland expression vector that contained the goat β-casein gene pro-moter, 5’upstream regulatory region, exons 1, 2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F0 mice were obtained. Of the 33, 8 mice (5 , 3 ) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.
文摘Due to the functional importance of bovine milk protein polymorphisms, their correct discrimination is of great interest both from a scientific and practical point of view. Nowadays a large number of commercial platforms are available for semiautomated or fully automated SNP geno-typing. However, in some cases the use of simple and rather cheap methods is an effective tool to be implemented within one’s own laboratory for the routine analysis of a specific SNP. The present paper describes two simple tests based on the bidirectional allele-specific polymerase chain reaction (BAS-PCR) developed for the identification of β-casein (CSN2) B and I genetic variants. The practical application of the two methods on a panel of 84 Italian Brown bulls and 100 Italian Friesian cows is also discussed, including the biological significance of the two genetic variants and the importance of taking their occurrence into account when linkage analyses are performed on milk functional properties. A combined system for analysing milk protein variants by isoelectrofocusing (IEF) and the BAS-PCR assay developed for CSN2*I is described.
文摘Cationic peptide with the sequence INKKI 41-45 was isolated from bovine β-casein after tryptic hydrolysis and synthetized. The aim of this work was to evaluate the antiproliferative activity in vitro and antitumor effect in animal model. The in vitro cytotoxicity was evaluated on B16F10 melanoma cells by MTT assay. Detection of apoptosis was measured using the annexin V/PI double staining and cell cycle analysis performed flow cytometry. Caspase-3 activity was analyzed with substrate specific fluorogenic DEVD-MCA. In vivo, antitumor activity was evaluated in B16F10 melanoma tumor-bearing C57BL/6J mice. The animals were treated with 55 mg/kg INKKI administered into peritumoral region, while control group received saline solution. The following antitumor parameters were examined: tumor volume, number of metastases, tumor delayed time, tumor doubling time. Histological analyses were performed with H & E staining. The results showed that INKKI induced dose-response cytotoxicity selective for B16F10 melanoma cells (IC50 1.7 μM) and did not present cytotoxic effects for FN1 fibroblast cells. INKKI-induced apoptosis detected trough of annexin V/PI assay and it was accompanied with an increase of sub-G1 apoptotic fractions and significant increase of caspase-3 cleavage. The tumor-bearing mice treated with INKKI showed a significant reduction in tumor volume of 72.62% and decreased of metastasis number loci. In addition, INKKI caused a significant delay in tumor growth and prolonged the tumor doubling time. Histological analysis revealed an increased of necrosis areas and reduction of tumor cells in tumor treated with INKKI, it was a many hallmark of its antitumor effects observed from in vivo experiments. In conclusion, we show that INKKI is a peptide that could be considered a new putative candidate development to anticancer therapy drug.
